Samuel Waxman

The isolation and characterization of the folate binding protein from goat milk

It is now known that there are specific folate binding proteins (FABP) in various tissues such as serum, milk and intestinal mucosa [2,3,4]. Whereas the FABP have been purified from cow milk [5], human milk [6] and umbilical cord serum [7] the study of the physical characteristics and physiologic functions has been hampered by the small quantities of FABP present. Goat milk, which contains a low concentration of folic acid, was recently reported to contain high levels of unsaturated FABP [8]. Affinity chromatography has been used successfully to separate the FABP from large quantities of goat milk and the present communication describes its physical and immunological characteristics.

The Role of Folic Acid Binding Proteins (FABP) in the Cellular Uptake of Folates

Summary HeLa cells and human lymphocytes were grown in complete media or conditioned in folate deficient media and were used to study the physiology of folic acid binding proteins (FABP). The uptake of tritiated folic acid (3HPGA) and N-5-methyl tetrahydrofolic acid (3H methyl-THFA) by folate replete HeLa cells increased for 3 hr, was temperature dependent, was greater for 3H methyl-THFA than 3HPGA and was not influenced by preincubation with Dilantin or ethanol. HeLa cells conditioned in folate deficient media after 1 week exhibited deranged DNA synthesis (abnormal deoxyuridine suppression of 3H thymidine into DNA and a decreased growth curve) which was progressive with degree of folate deficiency. The uptake of 3HPGA and 3H methyl-THFA was greater (five and three times, respectively) in the folate deficient HeLa cells than in the folate replete HeLa cells. 3HPGA or 3H methyl-THFA bound to the FABP of folate deficient sera, some uremic sera, human or cows milk was less available for the uptake by the HeLa cells. The percent uptake of 3HPGA by HeLa cells was inversely related to the amount of FABP in several sera tested. FABP added to the culture medium following a 1 hr pulse of 3HPGA did not decrease the cellular uptake of 3HPGA. Evidence was found for an energy dependent, active mechanism for efflux of 3HPGA from the HeLa cell.

Uptake of Tritiated Folates by Human Bone Marrow Cells in Vitro

Summary. Using incubation periods up to 4 hr, it was demonstrated that uptake of tritiated pteroylglutamic acid ([3H]PteGlu) by human bone marrow cells in vitro was in the range of six‐fold greater than uptake by reticulocytes. Uptake was temperature‐dependent, increasing during 4 hr at 37°C but not at 4°C; a similar temperature dependence was found for the uptake of [3H]methyltetrahydrofolate ([3H‐CH3]H4PteGlu). The pH optimum for [3H]PteGlu uptake was in the range of 7.4. Percentage uptake decreased as concentration of [3H]PteGlu increased. Preincubation with unlabelled PteGlu reduced uptake of subsequently added [3H]PteGlu by twice as much as did preincubation with methotrexate, suggesting that methotrexate may only partly share the uptake mechanism for [3H]PteGlu. [3H]PteGlu uptake was not affected by preincubation with diphenylhydantoin or a sulphydryl inhibitor. Uptake of [3H‐CH3]H4PteGlu by human bone marrow cells appeared to be approximately twice the uptake of [3H]PteGlu. The findings support the concept of two mechanisms for folate uptake by human reticulocytes and bone marrow cells: an energy‐dependent, active carrier mechanism probably of primary physiologic significance, and a seemingly passive diffusion‐like mechanism, probably primarily of pharmacologic significance.

The isolation of the folate binding protein from commercially purified bovine beta lactoglobulin

Commercially prepared bovine beta lactoglobulin contains binding determinants for both reduced and oxidized mono and polyglutamates [2] and therefore has been utilized as a stable binder in the radioisotopic assays for serum and whole blood folates [3,4]. However, because of the comparatively large amount of the crystalline product needed to bind 50% of a tracer amount of tritiated pteroylglutamic acid ( [3 H] PGA), (0.25 ng [3 HI PGA bound/0.1 mg) it was felt that the folate binding moiety was not the beta lactoglobulin per se but rather a separate protein purifying very closely to the beta lactoglobulin itself. As part of a study to identify the folate binding protein (FABP) of cows milk we fractionated mixed beta lactoglobulin into its phenotypic alleles and in so doing effectively separated the folate binder from the composite whole. The present report describes the isolation of FABP from the beta lactoglobulin fraction of cows milk in sufficient quantity to enable its preliminary characterization.

Measurement of Red Cell Folate Levels by 3H-Pteroylglutamic Acid (3H-PteGlu) Radioassay

Summary. A siniple, rapid radioassay is described for the determination of red cell folate activity utilizing commercially available beta lactoglobulin as thc folate binding protein. The standard curve is prepared with a pteroylpolyglutamate and 3H‐pteroylglutamic acid. The test is performed with a maximum of 20 nicrolitres of whole blood and multiple samples can be processed within 1 hr. Values of red cell folate in normal subjects range from 200 to 875 ng/ml packed cells giving good correlation with established microbiological procedures. Red cell folate levels were lowest in patients whose folate deficiency was accompanied by anaemia. This assay may be useful for measurement of pteroylpolyglutamates in other tissues since an equimolar amount of N‐5‐methyl tetrahydrofolate is barely measurable under the conditions of this assay.

Characteristics of a Novel Serum Vitamin‐B12‐Binding Protein Associated with Hepatocellular Carcinoma

Summary. A novel protein designated as hepatoma B12 binder (HBB), the predominant vitamin‐B12‐binding protein in the serum of two adolescents with hepatocellular carcinoma, has been characterized. HBB is more acidic and has different electrophoretic mobility and elution behaviour on gel filtration than normal or chronic myeloid leukaemia transcobalamin I. HBB is a member of the family of‘R’type vitamin‐B12‐binding proteins since it immunologically cross reacts with anti‐R antibody and does not deliver vitamin B12 to the cell. HBB is unique in that it is tumour‐related, elaborated in enormous quantity and possesses physical characteristics best explained by a different carbohydrate composition from that of transcobalamin I.

The Significance of Folate Binding Proteins in Folate Metabolism

Various aspects of folate metabolism in the microorganism and in the multicellular organism call for functional, specific folate binding proteins. These folate binding protein functions would include extracellular, intercellular, and intracellular folate transport, distribution, and storage.

Phosphate-induced phosphoribosylpyrophosphate elevations to assess deranged folate and purine nucleotide metabolism.

Abstract Phosphoribosylpyrophosphate (PRPP) levels increase several-fold in HL-60 cells adapted to folate deficiency either by continuous passage in folate-deficient medium or by short-term incubation with 10−8 M methotrexate (MTX). The addition of folic acid (PteGlu) or 5-formyltetrahydrofolic acid (5-CHO-H4PteGlu) in the form of Leucovorin normalizes this effect. The reactions for measuring PRPP levels are time and temperature dependent and are influenced by PRPP-reacting substances in undialyzed serum. Inorganic phosphate (PO4), when added to the assay, markedly stimulates PRPP levels in HL-60 cells and can be used to stress folate-dependent PRPP utilization for purine synthesis. The integrity of the folate-dependent pathways of purine-synthesizing cells can be sensitively assessed by measurement of PRPP levels during a 2-hr assay in the presence of PO4 in medium free of folate but containing dialyzed serum. In HL-60 cells that are folate deficient or in the presence of MTX (as low as 2 × 10−9 M), PO4-stimulated PRPP levels remain elevated due to ineffective utilization unless folate is added to the incubation mixture. The sensitivity of this PRPP assay to metabolically assess the integrity of folate-dependent reactions in purine synthesis is comparable to that of the deoxyuridine suppression assay. Inorganic phosphate can also be used to stimulate the incorporation of purine analogs, such as 6-mercaptopurine, into intact red blood cells which may have therapeutic implications for targeting drug delivery.

EVOLUTIONARY SIGNIFICANCE OF PERSISTENCE OF LATENT ONCOGENIC VIRUS INFORMATION IN VERTEBRATES

Abstract A hypothesis relating the independent schools of cancer research—virological and immunological—is proposed. It is suggested that oncogenic-virus information is a stable integral part of normal D.N.A. in most vertebrate species, including man, and that this information normally benefits the species, rather than doing harm, as is generally suggested for oncogenic viruses. Transcription of R.N.A. tumour viruses by derepression of such genetic information by carcinogenic agents leads to new antigenic information on the surfaces of transformed cells. These new antigens lead to recognition and rejection of the abnormal cell by the hosts cellular immune system.

The proportions of hemoglobin types induced in mouse erythroleukemia cells vary with the inducer or combination of inducers, the inducer concentration and the time of induction.

The relative amounts of hemoglobin (Hb) major and Hb minor accumulated during induction of erythrodifferentiation in mouse erythroleukemia (MEL) cells were studied. The ratio of major to minor was found to depend not only upon the inducer tested (as reported previously by others), but also upon the concentration of the inducer and the time of exposure to the inducer as well as the specific cell line of MEL cells studied. At concentrations required for optimal induction of differentiation, certain agents led to the accumulation of predominantly Hb major, but suboptimal concentrations of the same inducers led to predominantly Hb minor accumulation. After a relatively short induction time (2 da) utilizing a given inducer either the level of Hb minor was higher than that of Hb major or the levels of the two Hbs were approximately equal, but after longer induction periods (3-7 da) Hb major was more abundant than Hb minor. In addition, it was found that the three proteases tested induced predominantly Hb minor. The addition of suboptimal concentrations of low molecular weight inducers acted synergistically with a given protease to produce a high yield of Hb-containing cells. When these agents were added singly they induced relatively low Hb major/Hb minor ratios, but when a low molecular weight inducer was added together with a protease in a synergistic combination, elevated ratios were induced. The proportions of hemoglobin types induced in MEL cells may be related in part to the intensity of the induction response. In view of these data, classifications of inducers based solely on the ratios of Hb types produced must be guarded.